Pivotal Role of the C-terminal DW-motif in Mediating Inhibition of Pyruvate Dehydrogenase Kinase 2 by Dichloroacetate

Abstract

The mitochondrial pyruvate dehydrogenase complex (PDC) is down-regulated by phosphorylation catalyzed by pyruvate dehydrogenase kinase (PDK) isoforms 1-4. Overexpression of PDK isoforms and therefore reduced PDC activity prevails in cancer and diabetes. In the present study, we investigated the role of the invariant C-terminal DW-motif in inhibition of human PDK2 by dichloroacetate (DCA). Substitutions were made in the DW-motif (Asp-382 and Trp-383) and its interacting residues (Tyr-145 and Arg-149) in the other subunit of PDK2 homodimer. Single and double mutants show 20 -60% residual activities that are not stimulated by the PDC core. The R149A and Y145F/R149A mutants show drastic increases in apparent IC 50 values for DCA, whereas binding affinities for DCA are comparable with wild-type PDK2. Both R149A and Y145F variants exhibit increased similar affinities for ADP and ATP, mimicking the effects of DCA. The R149A and the DWmotif mutations (D382A/W383A) forestall binding of the lipoyl domain of PDC to these mutants, analogous to wild-type PDK2 in the presence of DCA and ADP. In contrast, the binding of a dihydrolipoamide mimetic AZD7545 is largely unaffected in these PDK2 variants. Our results illuminate the pivotal role of the DW-motif in mediating communications between the DCA-, the nucleotide-, and the lipoyl domain-binding sites. This signaling network locks PDK2 in the inactive closed conformation, which is in equilibrium with the active open conformation without DCA and ADP. These results implicate the DWmotif anchoring site as a drug target for the inhibition of aberrant PDK activity in cancer and diabetes.The pyruvate dehydrogenase complex (PDC) 2 catalyzes the oxidative decarboxylation of pyruvate to produce acetyl-CoA, linking glycolysis to the Krebs cycle (1-3). The PDC is a 9.5-megadalton catalytic machine comprising multiple copies of the three catalytic components pyruvate dehydrogenase (E1p), dihydrolipoyl transacetylase (E2p), and dihydrolipoamide dehydrogenase (E3); as well as the two regulatory enzymes pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase. The PDC is organized around a structural core comprising multiple subunits of E2p and a non-catalytic component that specifically binds E3 (i.e. the E3-binding protein (E3BP)). Each E2p subunit contains two consecutive N-terminal lipoic acid-bearing domains, termed L1 and L2 (beginning from the N terminus), followed by the E1p-binding domain and the C-terminal inner core/catalytic domain, with these independent domains connected by unstructured linker regions. By analogy, each E3BP subunit consists of a single N-terminal lipoic acid-bearing domain (referred to as L3), the E3-binding domain, and the non-catalytic inner core domain. Together, the inner core domains of E2p and E3BP assemble to form the pentagonal dodecahedral 60-meric E2p/E3BP core.The mammalian PDC is tightly regulated by reversible phosphorylation. The phosphorylation of E1p by PDK isoforms inactivates the PDC, whereas dephosphorylation by pyruvate dehydroge...

Mitochondrial activation by inhibition of PDKII suppresses HIF1a signaling and angiogenesis in cancer

“…72 Very recently, PDKII and DCA were crystallized together revealing molecular interactions that explain the relatively specific inhibition of PDKII by DCA. 73,74 Our data using siRNA to inhibit PDKII and PDH in this and our previous work 7 support the fact that DCA works primarily by inhibition of PDKII. It is possible, however, that after chronic DCA therapy PDKII may be downregulated, or compensated by PDK isoforms that are less sensitive to DCA.…”

Inactivation of Pyruvate Dehydrogenase Kinase 2 by Mitochondrial Reactive Oxygen Species

“…Cys-392 is located on the flexible C-terminal tail of PDHK2 next to a conserved DW-motif that is required for partial ordering of the C-terminal tails (Fig. 7A) (26). Oxidative modification of Cys-392 could prevent formation of the L2 binding site, whereas the C392A mutation could allow association of PDHK2 with L2 even under oxidizing conditions (Fig.…”

Furoates and thenoates inhibit pyruvate dehydrogenase kinase 2 allosterically by binding to its pyruvate regulatory site

“…This experiment demonstrated that both DCA and 8 bind to the same pocket of the enzyme. Given that DCA is a bona fide pyruvate-site inhibitor [10–12], we conclude that, as predicted by the computational studies, 8 exerts its inhibitory action by binding to the allosteric pyruvate regulatory site of the enzyme.…”

“…Synthetic PDK inhibitors have been discovered for all four binding sites that are large enough to host small organic molecules [10–16]. However, dichloroacetic acid (DCA) remained to date the only bona fide pyruvate-site PDK synthetic inhibitor [10–12]. …”